In 1976 it was reported that a discrete lipoprotein fraction isolated from normal human plasma inhibited mitogen- and allogenic cell-stimulated human lymphocyte proliferation in vitro (Curtiss et al., J. Immunol., 116:1452, (1976)). This inhibitory plasma lipoprotein was termed LDL-In for Low Density Lipoprotein-Inhibitor because the active fraction is localized to a less dense subfraction of total LDL of density 1.006-1.063 g/ml. The characteristics of LDL-In-mediated inhibition in vitro are as follows: LDL-In has comparable inhibitory activity for phytohemagglutinin (PHA), pokeweed mitogen (PWM), and allogenic cell-stimulated human lymphocyte proliferation. The inhibitory activity of LDL-In is non-toxic and independent of mitogen concentration. Suppression by LDL-In is time dependent and approximately 18 hr of exposure of the lipoprotein to the lymphocytes before stimulation is required for maximum induction of a, stable suppressed state. LDL-In does not inhibit .sup.3 H-thymidine uptake when it is added to the cultures 18-20 hr after stimulation, suggesting that this lipoprotein influences metabolic events associated with an early inductive phase of lymphocyte activation.
The immunosuppressive activity of LDL-In has been studied in a number of systems both in vitro and in vivo. To summarize, in vitro activities of LDL-In include suppression of: a) mitogen stimulated .sup.3 H-thymidine uptake, Curtiss et al., J. Immunol., 116:1452, (1976), b) allogenic cell-stimulated .sup.3 H-thymidine uptake (Curtiss et al., J. Immunol., 116:1452, (1976), Curtiss et al., J. Immunol., 118:1966, (1977) , c) the primary generation of cytotoxic T cells (Edgington et al., Regulatory Mechanisms in Lymphocyte Activation: Proceedings of the Eleventh Leukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York, pp. 736, (1977)), d) pokeweed mitogen stimulated immunoglobulin synthesis (Curtiss et al., J. Clin. Invest., 63:193, (1979)), and e) B-cell Epstein Barr Virus transformation (Chisari et al., J. Clin. lnvest., 68:329, (1981)). In vivo LDL-In has been shown to inhibit: a) the primary humoral immune response to sheep red blood cells (Curtiss et al., J. Immunol., 118:648, (1977), DeHeer et al., Immunopharmacology, 2:9, (1979), Curtiss et al., Cell. Immunol., 49:1, (1980)), b) the primary generation of cytotoxic T-cells (Edgington et al., Regulatory Mechanisms in Lymphocyte Activation: Proceedings of the Eleventh Leukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York, pp. 736, (1977)), and c) immunologic attention of tumor growth (Edgington et al., Cancer Res., 41:3786, (1981), Edgington et al., Dietary Fats and Health., ACOS Monograph No. 10, Perkins and Visek, eds., pp. 901, (1981)).
The effects of lipoproteins on immune cell function in vivo are exceedingly complex. A major finding of the investigation of the physiologic implications of immunosuppression by LDL-In in vivo is that the observed functional outcome is strikingly dose dependent. This important concept is best illustrated by describing in more detail studies of the effects of LDL-In on the survival of experimental animals challenged with syngeneic tumors (Edgington et al., Cancer Res., 41:3786, (1981), Edgington et al., Dietary Fats and Health., ACOS Monograph No. 10, Perkins and Visek, eds., pp. 901, (1981)). Seemingly divergent effects of LDL-In are observed on the growth of the syngeneic SaD2 fibrosarcoma in DBA/2 mice. The growth of 1.times.10.sup.5 viable tumor cells in control mice without immunoprotection (i.e., 10-days prior immunization with 10.sup.-6 irradiated tumor cells) is detectable at 25 days and proceeds rapidly until death at about 43 days. In contrast, tumor growth is slower in immunoprotected mice. This tumor growth is characterized by a reduction in tumor mass of at least a half and no deaths by day 60. Intravenous administration of high doses of LDL-In 24 hr before immunoprotection with killed tumor cells abolishes the protective effect of immunization. This dose corresponds to a dose that is required to abolish both B-cell and T-cell effector cell functions. The administration of an intermediate dose of LDL-In before immunoprotection with the killed tumor cells has no discernable effect on the subsequent growth of the viable tumor cell challenge. In contrast, intravenous administration of even lower doses of LDL-In 24 hr before immunoprotection with killed tumor cells results in the enhancement of tumor rejection and host survival. This dose of LDL-In is concordant with the dose required for selective inhibition of suppressor cell function in vitro (Curtiss et al., J. Clin. Invest., 63:193, (1979)). Thus, depending upon the amount of immunoregulatory lipoprotein that a particular lymphocyte population is exposed to in vivo, very different functional outcomes will result.
Lipoproteins are cleared from the plasma by binding to high-affinity receptors on liver cells and extrahepatic tissues such as the adrenal glands and ovaries (Kowal, R.C. et al., Proc. Natl. Acad. Sci. USA, 86:5810-5814, (1989). Two distinct sets of receptors bind APO E-containing lipoproteins. The low density lipoprotein (LDL) receptor [Yamamoto et al., Cell, 39:27-38 (1984)], 70% of which are thought to be located on hepatic cells, binds very low density lipoproteins (VLDL) and Apo E-containing remnants of chylomicrons. The existence of a second set of LDL-receptors, termed "remnant receptors", is inferred from studies showing that the plasma clearance of APO E-containing chylomicron remnants occurs at normal rates in animals with genetically defective LDL-receptors. Recently, an LDL-receptor-related protein (LRP) has been found on the surface of hepatic cells. Herz et al., EMBO, 7:4119-4127 (1988). LRP shares cysteine-repeat sequences with LDL and has been shown to bind and mediate the extracellular clearance of APO E-containing lipoproteins (Kowal, R.C. et al. Proc. Natl. Acad. Sci. USA, 86:5810-5814, (1989).
Plasma lipoproteins differ from most humoral immunoregulatory molecules in that they are large heterogenous non-covalent complexes of lipid and protein. An important step to understanding the mechanism of lipoprotein regulation of cell function is an identification of the constituent(s) of the lipoprotein particle that mediate the observed biologic effects. Plasma lipoproteins contain various amounts of apoproteins, glyceride, free and esterified cholesterol, phospholipid, glycolipid and free fatty acid. Many of these constituents of lipoproteins can by themselves influence cell function.
The heterogeneity of apoproteins in terms of structure, exposure and function make them likely candidates as biologically important constituents of LDL-In, and the contribution of the apoproteins to biologic activity has been extensively studied. LDL-In contains Apo B, Apo E, and each of the C apoproteins. The specific role played by Apo B and Apo E in LDL-In was investigated immunochemically using Apo B-specific and Apo E-specific monoclonal antibodies (Curtiss et al., Fed. Proc., 40:348, (1981), Curtiss et al., Atherosclerosis, 2(5):A111, (1982)). Some, but not all, of the Apo B-antibodies and each of the Apo E-specific antibodies bind and facilitate the indirect precipitation and removal of the inhibitory activity of LDL-In from a lipoprotein fraction. These results indicate that LDL-In contains both apoproteins B and E, but they do not identify which apoprotein is important to or required for activity.
Further substantiation that Apo E and Apo B-containing lipoproteins are important regulators of lymphocyte function has come from studies of the inhibitory properties of fetal cord blood plasma lipoproteins (Curtiss et al., J. Immunol., 133:1379, (1984)). In these studies a direct correlation between Apo E and inhibition was established. Cord blood lipoprotein concentrations are lower than those of adult, i.e., the low density lipoprotein (LDL) level in cord blood is 30% that of adult, whereas the high density lipoprotein (HDL) level is 50% of adult levels. In contrast, the Apo E concentration in fetal cord blood is 2-fold higher than adult (Curtiss et al., J. Immunol., 133:1379, (1984)). Therefore, the capacity of LDL and HDL to inhibit mitogen-stimulated .sup.3 H-thymidine uptake in adult peripheral blood mononuclear cells was used as an in vitro system to study immunosuppression. Relative to adult lipoproteins, cord blood LDL and HDL are 2 to 4 times more potent in inhibiting cellular proliferation. Radioimmunoassay results demonstrate a strong correlation between the amount of Apo E in cord blood LDL and HDL and the inhibition of cell proliferation. Furthermore, selective removal of Apo E-containing lipoproteins decreases the inhibitory capacity of cord blood LDL and eliminates almost completely inhibition by HDL. The results indicate that cord blood lipoproteins containing Apo E in association with either LDL or HDL can suppress the immune response (Curtiss et al., J. Immunol., 133:1379, (1984)). The fetus is an allograft to its mother. Therefore the relatively high fetal levels of Apo E may have functional significance in the establishment of self as well as maintenance of the fetus in utero.
More recently, the inhibitory activity of isolated (lipid-free) Apo E has been studied. Immunosuppression was measured as inhibition of .sup.3 H-thymidine uptake by peripheral blood mononuclear cells (PBM) with phytohemagglutinin (PHA). Apo E isolated from lipoproteins had good activity (i.e., approximately 15 ug/ml was required for 50% inhibition, and maximal inhibition occurred at 20 ug/ml), whereas fractions containing the lipid-free C apoproteins were not inhibitory at&gt;20 ug/ml (Pepe et al., J. Immunol., 126:3716, 1986)). Suppression of lymphocyte proliferation by the native lipoprotein, LDL-In, is irreversible and has distinguishable temporal requirements (Curtiss et al., J. Immunol., 116:1452, (1976), Curtiss et al., J. Immunol., 118:1966, (1977)). Suppression by isolated Apo E is identical. That is, cells exposed to isolated Apo E for 24 hr and washed free of non-cell associated Apo E before mitogen stimulation, remain fully suppressed. And, maximal inhibition is obtained with either LDL-In or Apo E only after a 24 hr exposure of the cells before the addition of mitogen. Exposure periods of 18 hr or less result in little or no suppression by either inhibitor. Furthermore, cells receiving inhibitors or PHA simultaneously, or cells receiving either inhibitor after PHA exposure, are fully capable of responding to mitogen induction, suggesting that neither LDL-In nor Apo E are directly toxic. The irreversibility and temporal requirements of suppression confirm that Apo E isolated from lipoproteins has the same characteristics of immunosuppression as LDL-In and that an active moiety of LDL-In is Apo E (Pepe et al., J. Immunol., 126:3716, (1986)).
Cardin et al., Biochem. Biophys Res. Comm., 154:741-745 (1988) reported that a polypeptide portion of Apo E having an amino acid residue sequence identical to that of Apo E residues 141-155 inhibits lymphocyte proliferation when coupled to bovine serum albumin (BSA). However, conspicuously absent from the study of Cardin et al. was any control for cell viability allowing for a determination of whether or not the inhibition observed was due to cytotoxicity of the peptide-BSA conjugate.
By way of further background, Dyer et al., J. Biol. Chem., 263:10965-10973 (1988) reported that isolated lipid free rat Apo inhibits androgen production by the ovarian theca and interstitial cells induced by the gonadotropin, luteinizing hormone (LH).